The phosphoinositide-3 kinase (PI3K) pathway regulates critical cellular functions such as cell cycle progression, growth, survival, and differentiation as well as the metabolic actions of insulin (Hennessy et al. Nature Rev. Drug Dis. 4:988-1004 (2005)). Modulation of the activity of kinases downstream of PI3K is mediated by 3-phosphoinositide dependent kinase 1 (PDK1), a 63-kD serine/threonine kinase that is ubiquitously expressed in human tissues (Storz and Toker, Front. Biosci. 7:886-902 (2002). PDK1 contains an amino-terminal kinase domain, a linker region and a pleckstrin-homology (PH) domain at the carboxyl-terminus. The PH domain binds to the lipid products of PI3K (phosphatidylinositol 3,4,5-triphosphate, PIP3) with high affinity, and facilitates co-localization of the kinase with its PH-domain-containing substrates. Substrates of PDK1 include many of the AGC family of protein kinases (the cAMP-dependent, cGMP-dependent, and protein kinase C), including AKT/PKB, p70S6K, cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC), serum and glucocorticoid-inducible kinase (SGK), p90 ribosomal protein kinase (RSK), p21-activated kinase-1 (PAK1), PRK1/2, and others (Wick and Liu, Curr. Drug Targets Immune Endocr. Metabol. Disord. 1:209-221 (2001); Mora et al., Semin. Cell Dev. Biol. 15:161-170 (2004)). Recent in vivo studies with PDK1(−/−) and PDK1(−/+) mice showed that AKT, p70S6K, RSK and protein kinase C are key mediators of PDK1 function, regulating diverse cellular functions (Lawlor et al EMBO J. 21:3728-3738 (2002); Williams et al., Curr. Biol. 10:439-448 (2000); Storz and Toker, Front. Biosci. 7:886-902 (2002)). Activation of these substrates by PDK1 leads to an increase in glucose uptake, protein synthesis, and inhibition of pro-apoptotic proteins.
Dysregulation of the PI-3 kinase pathway is seen in a variety of cancers. A significant number of cancers possess mutations in genes that result in elevation of cellular levels of PIP3. Increased levels of PIP3 leads to activation of AKT and p70S6K kinases, which promote the proliferation and enhanced survival of these tumor cells. For example, genetic alterations of the PI3K gene including amplifications and activating mutations have been observed in various cancers (Hennessey et al., Nature Rev. Cancer 4, 988-1004 (2005)). One of the most common mutations giving rise to elevated levels of PIP3 is in the PIP3 3-phosphatase PTEN gene (Leslie and Downes, Cell Signal. 14:285-295 (2002); Cantley, Science 296:1655-1657 (2002)). Germline mutations of PTEN are responsible for human cancer syndromes such as Cowden disease (Liaw et al., Nature Genetics 16:64-67 (1997)). PTEN is deleted in a large percentage of human tumors and tumor cell lines without functional PTEN show elevated levels of activated AKT (Li et al. supra, Guldberg et al., Cancer Research 57:3660-3663 (1997), Risinger et al., Cancer Research 57:4736-4738 (1997)).
AKT/PKB (consisting of 3 family members, AKT1, AKT2, AKT3) is a substrate for PDK1 and is an important mediator of the physiological effects of insulin and several growth factors including EGF, PDGF, and IGF-1 (Manning and Cantley, Cell 129:1261-1274 (2007)). AKT is activated by phosphorylation events occurring in response to PI3K signaling. PI3K phosphorylates membrane inositol phospholipids, generating the second messengers PIP3 and phosphatidylinositol 3,4-diphosphate, which have been shown to bind to the PH domain of AKT. The current model of AKT activation proposes recruitment of the enzyme to the membrane by PIP3 through the PH domain of PDK1. Co-localization of AKT and PDK1 at the membrane allows for AKT modification and activation by PDK1 and possibly other kinases (Hemmings, Science 275:628-630 (1997); Hemmings, Science 276:534 (1997); Downward, Science 279:673-674 (1998)). Phosphorylation of AKT1 occurs on two regulatory sites, Thr308 by PDK1 in the catalytic domain activation loop and Ser473 (most probably by TORC2 mTOR complex) near the carboxy terminus (Alessi et al., EMBO J. 15:6541-6551 (1996); Meier et al., J. Biol. Chem. 272:30491-30497 (1997)). Analysis of AKT levels in human tumors revealed that AKT is overexpressed in a significant number of ovarian (Cheung et al., Proc. Natl. Acad. Sci. U.S.A. 89:9267-9271 (1992)) and pancreatic cancers (Cheung et al., Proc. Natl. Acad. Sci. U.S.A. 93:3636-3641 (1996)). AKT was also found to be overexpressed in breast and prostate cancer cell lines (Nakatani et al. J. Biol. Chem. 274:21528-21532 (1999)). More recently, a transforming mutation in the PH domain of AKT1 was detected in human breast, colorectal and ovarian cancers (Carpten et al., Nature 448:439-444 (2007)). Specific inhibitors of PI3K or dominant negative AKT mutants abolish survival-promoting activities of growth factors or cytokines. It has been previously described that inhibitors of PI3K (LY294002 or wortmannin) blocked the activation of AKT. In addition, introduction of constitutively active PI3K or AKT mutants promotes cell survival under conditions in which cells normally undergo apoptotic cell death (Kulik et al., Mol. Cell Biol. 17(3):1595-1606 (1997); Dudek et al, Science 275(5300):661-665 (1997)).
PDK1 modulates affects the activity of a variety of substrates besides AKT. These substrates lack the PH domain seen in AKT and are therefore not dependent on co-localization with PDK1 on cell membranes. Important PH-domain-independent substrates of PDK1 are PKC, RSK and p70 S6K. RSKs have been recently implicated in promoting FGFR3-mediated hematopoetic transformation (Kang et al., Cancer Cell 12:201-214 (2007)). PDK1 activates RSK by phosphorylating its amino terminal kinase domain in an ERK-dependent manner (Cohen et al., Nature Chem. Biol. 3(3):156-160 (2007)). Also, recent studies revealed additional roles of PDK1 that could be important during tumorigenesis and metastasis, such as cell motility and migration (Primo et al., J. Cell Biol. 176(7):1035-1047 (2007); Pinner and Sahai, Nature Cell Biol. 10(2):127-137 (2008)).
Taken together, these observations suggest a beneficial role for an inhibitor of PDK1 in the treatment of cancer cells. Consistent with this, a hypomorphic mutation of PDK1 suppresses tumorigenesis in PTEN+/− mice (Bayascas et al., Curr. Biol. 15, 1839-1846 (2005)). Furthermore, antisense-based reduction of PDK1 levels in tumor cells leads to decreased tumor cell proliferation and increased apoptosis (Flynn et al. Curr. Biol. 10: 1439-1442 (2000)), and small molecule kinase inhibitors of PDK1 inhibit the growth of tumors cells in vitro and in vivo (Feldman et al., J. Biol. Chem. 280: 19867-19874 (2005); Gopalsamy et al. (J. Med. Chem. 50, 5547-5549 (2007); Tamguney et al. Exp. Cell Res. 314:2299-2312 (2008)). Finally, since knockdown of PDK1 by siRNA was shown to sensitise breast cancer cells to tamoxifen (Irons et al., Biochem. J. 417:361-370 (2009)), inhibition of PDK1 may have a therapeutic benefit in combination with other anticancer treatments.
PDK1 is reported to be a mediator of T-cell activation through NF-kB activation (Lee et al. Science 308: 114-118 (2005)) and also a regulator of T-cell development (Hinton et. al. Nat. Immunol. 5(5), 539-545 (2004)). Literature data also suggests that a PDK1 inhibitor may be useful for the treatment of autoimmune disease and transplant rejection (e.g. Park et al. Nat. Immunol. 10(2), 158-666 (2009)).
Given the close association of PDK1 with the AKT and PI3K pathways, and inhibitor of PDK1 may have beneficial use in treating diseases related to metabolism and aging, for example through the downstream inhibition of S6K1 signaling (Selman et al. Science 326: 140-144 (2009)).
It is an object of the instant invention to provide novel compounds that are inhibitors of PDK1.
It is also an object of the present invention to provide pharmaceutical compositions that comprise a pharmaceutical carrier and compounds useful in the methods of the invention.
It is also an object of the present invention to provide a method for treating cancer, immune and metabolic diseases that comprises administering such inhibitors of PDK1 activity.